The decomposition of chemiluminescent chemical compounds to release electromagnetic, and especially optically detectable, energy--usually luminescence in the form of visible light--is well known and understood. The incorporation of such light emitting reactants in art-recognized immunoassays, chemical assays, nucleic acid probe assays and chemical/physical probe techniques as the means by which the analyte, a substance whose presence, amount or structure is being determined, is actually identified or quantified has assumed increasing importance in recent years, particularly with the advent of enzymatically-cleavable 1,2-dioxetanes; see, for example, copending Bronstein U.S. patent application Ser. No. 889,823, "Method of Detecting a Substance Using Enzymatically-Induced Decomposition of Dioxetanes", filed Jul. 24, 1986; Bronstein et al U.S. patent application Ser. No. 140,035, "Dioxetanes for Use in Assays", filed Dec. 31, 1987 and Edwards U.S. patent application Ser. No. 140,197 "Synthesis of 1,2-Dioxetanes and Intermediates Therefor", filed Dec. 31, 1987.
Reactions that produce chemiluminescence exemplify yet another instance in which the medium, although not the message, can determine the intensity of the message transmitted. Chemiluminescent compounds that, upon decomposition in substances such as moderately polar or polar aprotic organic solvents, e.g., n-butanol, acetonitrile, dimethylsulfoxide or dimethylformamide, produce fluorophores that in turn emit light of adequate intensity for easy detection and quantitation will produce light of considerably lessened intensity when decomposed in a polar protic environment, and especially in aqueous media. But since all biological systems are aqueous--indeed, man himself is 97% water--the need to enhance the intensity of light produced by chemiluminescent labels or substrates in immunoassays, nucleic acid probe assays, chemical/physical probe techniques and other bioassays is obvious. One way to provide such enhancement, of course, is to use expensive optical or electronic equipment: single photon counters, luminometers, scintillation counters, etc. The present invention provides a far less expensive yet equally effective way of providing the needed light enhancement in aqueous media, and in many cases provides enhanced light intensity to a degree which permits detection by simple, inexpensive means, such as with a camera, instead of with complex detection instruments.
The present invention also permits the detection of lesser concentrations of analytes using the same quantities of chemiluminescent chemical compounds used in hitherto-practiced methods, and concomitantly permits the use of lessened amounts of chemiluminescent chemical compounds to detect the same concentrations of analytes as compared to those necessary in hitherto-practiced methods. B practicing this invention the intensity of light emitted by fluorophore decomposition products of chemiluminescent chemical compounds can be enhanced by a factor of at least -0%, but usually at least tenfold and oftentimes by factors of at least 100 to 1,000,000 times the intensities obtainable in aqueous media using the same chemiluminescent compounds in hitherto-practiced methods.
While we do not wish to be bound by any theory or mechanism advanced to explain the operation of this invention, we believe that our enhancer substances act in a polar protic environment, such as an aqueous medium, to bind fluorophore-containing fragments resulting from the decomposition of a chemiluminescent chemical compound and maintain such fragments in a stabilized conformation, possibly by hydrophobic or ionic interaction, or both, between the enhancer substance and the fluorophorecontaining fragment. This we believe in turn inhibits the fluorophore from releasing all or a substantial part of its excitational energy through non-light emitting pathways: vibrational relaxation in which energy is emitted as heat rather than light, intersystem crossing to other lower energy states, or other such mechanisms.